1. Field of the Invention
This invention generally relates to methods for screening for inhibitors of HIV Rev function. Specifically, this invention relates to a method of screening for inhibitors of HIV Rev function using a nucleic acid construct comprising a reporter gene positioned in the construct such that expression of the reporter gene increases when Rev function is decreased.
2. Background Art
Acquired Immune Deficiency Syndrome (AIDS), a fatal human disease, is generally considered to be one of the more significant diseases to affect humankind, and has affected numerous individuals worldwide. Because this disease is so widespread and so destructive, there is a vast amount of research currently being undertaken to find new therapies and new drugs which may provide some assistance in combating this virus. One approach for drug therapy is to target viral proteins in an attempt to inhibit or halt viral replication.
One of the viral proteins that is required for viral replication is Rev. This protein is necessary for high-level production of the gag and env structural proteins. One hypothesis concerning regulation of HIV gene expression is that Rev facilitates transport and stability of unspliced or singly spliced RNAs from the nucleus to the cytoplasm where these RNAs are subsequently translated. (Knight et al. "Expression of the art/trs protein of HIV and study of its role in viral envelope synthesis" Science 236:837-840 (1987) and Malim et al. "The HIV-1 rev trans-activator acts through a structural target sequence to activate nuclear export of unspliced viral mRNA" Nature 338:254-257 (1989)). These observations, however, may be a result of the specific model systems utilized in those studies since Arrigo et al. found that Rev did not appear to affect the cytoplasmic level of singly spliced vif, vpr, or env/vpu 2 RNAs in lymphoid cells, but appeared to regulate the assembly of these transcripts into polysomes for translation. (Arrigo et al. "Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs" Genes and Develop. 5:808-819 (1991)). Therefore Rev may have more than one mode for regulating HIV gene expression. Whatever specific mechanism or mechanisms by which Rev acts, it is clear that Rev has a major role in HIV gene expression since Rev-deficient mutants of HIV are unable to produce infectious virions and trans-dominant Rev mutants are able to downregulate the activity of wild-type Rev. ("Textbook of AIDS Medicine" Ed. Broder et al. Pub. by Williams and Wilkins, Baltimore, Md. (1994) and Malim et al. "Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function" Cell 58:205-214 (1989)). Inhibition of Rev function is therefore a clear candidate for drug or inhibitor targeting for therapeutic and prophylactic purposes, but screening compounds or compositions for inhibition of Rev function requires an effective and practical assay.
Previously utilized assays for Rev function include filter binding assays, gel mobility (gel shift) assays, spectroscopic assays, and capture assays. (See, e.g., WO 92/05195). These assays are relatively tedious and expensive, generally insensitive, and labor intensive. These assays are also relatively unsuitable for large scale, high volume assays involving multiple tests.
Similarly, assays for evaluating Rev function comprising nucleic acid amplification techniques are also labor intensive, relatively expensive, and generally unsuitable for large scale, high volume assays. (See, e.g., Arrigo et al. "Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method" J. Virol. 63:4875-4881 (1989)). These assays have the additional drawback of being limited to monitoring nucleic acid levels which are not necessarily related to the activity of a gene product.
Assays utilizing reporter genes have also been used in Rev-related studies. For example, see Raghavendar et al. "Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene" J. Virol. 69:5167-5170 (1995), Cochrane et al. "Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression" J. Virol. 65:5305-5313 (1991), Shukia et al. "Human chromosome 6- and 11-encoded factors support human immunodeficiency virus type 1 function in A9 cells" J. Virol. 70:9064-9068 (1996), Schiller et al "Rapid complementation assay for anti-HIV-1 drug screening and analysis of envelope protein function" Aids Res. Hum. Retro. 8:1723-1731 (1992), and U.S. Pat. No. 5,534,408 "2-Deoxystreptamine aminoglycoside inhibition of HIV/Rev binding," all of which utilize a chloramphenicol aminotransferase (CAT) assay where the activity of the chloramphenicol aminotransferase is positively correlated to Rev function. Additionally, see Bahner et al. "Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes" J. Virol. 67:3199-3207 (1993) and Xiaobin et al. "Assay systems for HIV Rev function using chimeric gene constructions" V Int. Conf. on AIDS, Jun. 4-9, 1989, p. 1262, which utilize the Escherichia coli lacZ gene operatively linked to an HIV gene wherein the expression of the lacZ reporter gene is positively correlated to Rev function.
The present invention provides a novel method in which the expression of the reporter gene increases with an increasing inhibition of Rev function, thereby providing a method with significantly more sensitivity than previously available techniques. Additionally, the methods provided by the present invention have the capacity to discriminate between an effect which is not Rev-specific which might indirectly affect the level of the reporter activity and a Rev-specific effect. Therefore the methods provided herein represent an important improvement over the art by providing a much needed means to evaluate potential anti-Rev drugs as well as providing a method by which one can screen for the inhibition of a Rev responsive element and a method by which one can monitor the expression of HIV structural genes relative to HIV regulatory genes.